The discovery of the real-time PCR technique as it is used today was made possible by two important findings. First, the Taq polymerase has, apart from its polymerase activity, a 5′–3′ exonuclease activity.10
Second, dual-labeled fluorogenic oligonucleotide probes have been created which emit a fluorescent signal only upon cleavage, based on the principle of fluorescence resonance energy transfer.11
In the TaqMan assay (Applied Biosystems, Foster City, CA), which was the first real-time PCR assay developed, these two principles are combined. In this system a probe, the so-called TaqMan probe, is designed to anneal to the target sequence between the classical forward and reverse primers (Fig. 1). The probe is dually labeled, with a reporter fluorochrome (e.g., 6-carboxyfluorescein, or FAM) at one end and a quencher dye (e.g., 6-carboxy-tetramethyl-rhodamine, or TAMRA) at the 3′ end. Importantly, in its intact form, the fluorescence emission of the reporter dye will be absorbed by the quencher dye. The probe has a melting temperature (m
) approximately 10°C higher than the m
of the primers, in order to anneal to the amplicon during the extension phase of the PCR process (which is performed at 60°C). Consequently, the probe will be degraded during the extension phase by the 5′–3′ exonuclease activity of the Taq polymerase. This will result in an increase in reporter fluorescence emission because reporter and quencher are separated. The amount of fluorescence released is directly proportional to the amount of product generated in each PCR cycle and thus can be applied as a quantitative measure of PCR product formation.
FIGURE 1 Schematic representation of the TaqMan principle. A: Primers and probe anneal to the target gene. Fluorescence emission does not occur because the probe is still intact. B: During the extension phase of the PCR reaction, the probe is cleaved by the 5′–3′ (more ...)
As the technology has become more commonly used, other sophisticated chemistries have been developed to directly measure PCR product accumulation by fluorescence emission. Examples include molecular beacons, Scorpions, hybridization probes, and minor groove binder (MGB) probes (e.g., Eclipse, TaqMan MGB). Other new technologies include ResonSense probes, light-up probes, and Hy-Beacon probes.12, 13
Finally, the use of double-stranded DNA minor groove binding dyes, such as SYBR Green I, is a cheaper, widely used alternative, where the need for an expensive probe can be avoided.
One can choose among a diversity of competing instrumentations that have recently been launched on the market.12, 13
All of them run the PCR reaction as a closed tube and measure product accumulation in real time during the course of PCR amplification. Differences between the instrumentations are the sample format (tubes, microplates, strip tubes, capillaries, etc.), the maximum sample number (ranging from 16 to 384), the length of a run (ranging from 30 min to 2 h), the light source (halogen or laser), the fluorescence wavelength detection, the possibility of performing single or multiplex (i.e., measuring different fluorescence emissions simultaneously) PCR reactions, the availability of melting curve analysis, and finally the price. Some of the instruments are designed primarily for high-throughput applications, whereas others allow more flexibility. Therefore, the choice for an instrument will depend on the specific applications to be performed.
With any of the developed chemistries on any of the developed instrumentations, a software package is provided that measures the increase in fluorescence emission in real time, during the course of the reaction. This increase in fluorescence emission is directly related to the increase in target amplification. In the ABI Prism 7700 Sequence Detection System (SDS) (Applied Biosystems), for example, the software calculates a
Rn using the equation
Rn = Rn+
, with Rn+
being the fluorescence emission of the product at each time point and Rn−
being the fluorescence emission of the baseline.9
Rn values are plotted against cycle number, resulting in amplification plots for each sample (Fig. 2). Threshold values (Ct) are then determined as the cycle number at which the fluorescence emission (
Rn) exceeds a chosen threshold, which is usually 10 times the standard deviation of the baseline (this threshold level can, however, be changed manually if desired). Software will plot Ct values of unknown samples on the standard curve to determine the starting copy numbers of the unknown samples. Alternatively, the Ct values can be used as a direct quantitative measurement.
FIGURE 2 PCR amplification plot. Fluorescence emission is measured continuously during the PCR reaction and Rn (increase in fluorescence emission, from which the background fluorescence signal is subtracted) is plotted against cycle number. The threshold (more ...)